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PAPER
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Cite this: J. Mater. Chem. B, 2015, 3,
458
A simple strategy based on upconversion
nanoparticles for a fluorescent resonant energy
transfer biosensor†
Hao Zhu,a Yujie Ding,ab Anqi Wang,a Xu Sun,a Xing-Cai Wu*a and Jun-Jie Zhu*a
A novel aptasensor was fabricated for the detection of lysozyme and DNA based on the fluorescence
resonance energy transfer (FRET) technique between upconversion nanoparticles (UCNPs) and a dye
labeled aptamer. UCNPs can act as excellent emitters due to their low autofluorescence and high
penetration depth of biosamples. NaYF4:Yb, Er nanoparticles as UCNPs were synthesized and attached
with a dye labeled aptamer through a cationic polymer as an electrostatic linker to quench the
upconversion fluorescence intensity. The intensity can be restored after the addition of lysozyme or the
complementary DNA (target DNA) because of their strong interaction with the aptamer. The sensor
provided a linear concentration range from 30 to 210 nM for lysozyme and 40 to 200 nM for the target
Received 9th August 2014
Accepted 15th October 2014
DNA, the limit of detection was 2.5 nM and 2.8 nM, respectively. The sensor was also used to monitor
the lysozyme level in both human saliva and serum samples, and the results were consistent with the
DOI: 10.1039/c4tb01320d
reported values. The method was simple and convenient without the extra procedure of bioconjugation,
www.rsc.org/MaterialsB
and could be put to use for the determination of various targets in the future.
1. Introduction
In recent years, rare earth luminescent materials have shown
wide use in optical ber telecommunications, light-emitting
organic electroluminescence applications, lighting and other
areas of biomedicine. Among those materials, upconversion
nanoparticles (UCNPs) attract the most focus due to their
unique optical and chemical properties, such as sharp absorption and emission bands, high quantum yields, long lifetimes
and superior photostability.1–3 UCNPs are usually composed of
oxides, uorides, halogen oxides or other substrates as the
matrix and doped with trivalent rare earth ions (Er3+, Eu3+, Yb3+,
Tm3+, Ho3+ etc.) as the energy transfer system. UCNPs can
convert low-energy light (NIR or IR) to higher-energy light (UV or
visible) through multiple photon absorptions or energy transfers; as a result, the UCNPs exhibit low autouorescence from
biosamples and better penetration depth than the traditional
downconversion uorescent materials.4–8 Therefore, UCNPs can
be used as suitable candidates in sensing and bioimaging.
a
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and
Chemical Engineering, Nanjing University, Nanjing, 210093, P. R. China. E-mail:
[email protected]; [email protected]; Fax: +86-25-83597204; Tel: +86-2583597204
b
College of Biochemical Engineering, Anhui Polytechnic University, Wuhu, 241000,
P. R. China
† Electronic supplementary
10.1039/c4tb01320d
information
458 | J. Mater. Chem. B, 2015, 3, 458–464
(ESI)
available.
See
DOI:
Aptamers obtained with systematic evolution of ligands by
exponential enrichment (known as SELEX) are usually singlestranded DNA or RNA oligonucleotides.9 Aptamers can bind
specically to various target molecules, including amino
acids, antibiotics, peptides, vitamins, proteins and even
whole cells.10 In general, aptamers can be viewed as an ideal
replacement for antibodies because of their small size,
simple preparation, good stability, high reproducibility and
easy modication.11 Nowadays, a large number of analytical
approaches are being used to fabricate aptamer-based
sensors for protein detection, among which the uorescence
resonance energy transfer (FRET) process is oen adopted.12–16 For a well-designed FRET system, the donor and
acceptor can be brought to a proper distance from each other
exclusively through the recognition of target substances,
leading to a corresponding change in uorescence intensity.
Since Wang reported a FRET biosensor using UCNPs in
2005,17 UCNPs have been selected as energy donors in the
FRET system. Besides gold nanoparticles, uorescent dyes
and carbon materials have been used as energy acceptors. 18–24
Although inorganic materials are able to quench donors with
high efficiency, they also bring about instability in the
system. In addition, the UCNPs in the systems are oen
converted to the related oligonucleotides using cross-linkers
(EDC or DDC), which needs several complicated steps and
may decrease the efficiency.3,25 To simplify the assembly, it is
desirable to nd a novel strategy without the extra procedure
of bioconjugation.
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Paper
Journal of Materials Chemistry B
Lysozyme is a ubiquitous protein serving as a natural drug by
cleaving acetyl groups in the polysaccharide walls of bacteria
and exists universally in body tissues and secretions.26 The
enzyme has a molecular weight of about 14.3 kDa and an
isoelectric point value (pI) of 11.0. The abnormal levels of this
kind of enzyme in saliva, serum and urine have been associated
with many diseases, such as leukemia, meningitis, HIV and
renal diseases.27 Although some strategies have been reported
for lysozyme detection,26,28–32 only very few have been reported
for detection using UCNPs via the FRET technique.
Herein, we report a simple sensing platform for lysozyme
and the complementary DNA (target DNA) determination based
on FRET between uorescent UCNPs and a dye-labeled aptamer
through a cationic polymer as an electrostatic linker, which can
avoid the complicated covalent immobilization and modication of UCNPs.
2.3 Transferring hydrophobic OA-UCNPs to hydrophilic
PAA-UCNPs through ligand exchange
The strategy was developed via a modied literature procedure.34 Typically, 0.60 g of PAA was added to 12 mL diethylene
glycol (DEG) and the mixture was heated to 110 C with vigorous
stirring under a nitrogen atmosphere. A toluene solution of the
OA-UCNPs (30 mg of OA-UCNPs in 2 mL toluene) was added to
the system and then heated to 240 C and kept at the temperature for 1 h. Aer the solution was cooled down to room
temperature, excess dilute hydrochloric acid aqueous solution
(0.1 M) was added, and a white powder was obtained by
centrifuging. The powder was washed three times with pure
water and could be well dispersed into water and buffer
solution.
2.4 Loading PAH onto PAA-UCNPs for assembly of PAH–PAAUCNPs (ppUCNPs)
2.
2.1
Experimental section
Materials
All starting materials were obtained from commercial suppliers
and used as received. YCl3$6H2O (99.99%), YbCl3$6H2O
(99.99%), ErCl3$6H2O (99.9%), NaOH (98%), NH4F (98%),
1-octadecene (90%), oleic acid (90%), poly(acrylic acid) (PAA),
poly(allylamine hydrochloride) (PAH), lysozyme, thrombin and
bovine serum albumin (BSA) were purchased from SigmaAldrich. All oligonucleotides were supplied by Sangon Biotechnology Co., Ltd (Shanghai, China). Serum mixture specimens of
one hundred healthy persons were provided by the Affiliated
Drum Tower Hospital of Nanjing University. Other chemical
reagents of analytical grade were used directly without further
purication. Ultrapure water (Milli-Q, Millipore) was used
throughout. The sequences of oligonucleotides used in this
work are as follows:
Lysozyme aptamer: 50 -ATC AGG GCT AAA GAG TGC AGA GTT
ACT TAG-TAMRA-30
Target DNA: 50 -CTA AGT AAC TCT GCA CTC TTT AGC CCT
GAT-30
2.2
Synthesis of OA-UCNPs
The synthesis of NaYF4: 18% Yb, 2% Er nanoparticles was
developed via a modied literature procedure.33 In a typical
experiment, 1 mmol RECl3 (0.80 mmol YCl3, 0.18 mmol YbCl3
and 0.02 mmol ErCl3) was added into a 100 mL ask containing
7.5 mL oleic acid and 15 mL 1-octadecene. The solution was
heated to 160 C under a nitrogen atmosphere for 30 min and
then cooled down to room temperature. Thereaer, 10 mL
methanol solutions containing NH4F (4.0 mmol) and NaOH (2.5
mmol) were added into the solution and stirred for 30 min.
Aer the methanol evaporated, the solution was heated to
300 C under a nitrogen atmosphere for 1 h and cooled down to
room temperature. The resulting nanoparticles were precipitated by the addition of ethanol, collected by centrifugation,
washed with ethanol and water several times, and nally
redispersed in toluene.
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10 mg of the as-prepared PAA-UCNPs was redissolved in water
(pH 8.5) and 10 mg mL1 PAH solution (containing 20 mM
NaCl) was added to the system. The mixture was stirred for 1 h
at room temperature and the solid was collected by centrifuging
(16 000 rpm, 10 min), then the collected solid was repeatedly
washed with water to remove the unbounded polymer. The
obtained positively charged PAH–PAA-UCNPs (ppUCNPs) were
dispersed into Tris–HCl buffer (20 mM, containing 10 mM NaCl
and 10 mM MgCl2, pH 7.4) and used for subsequent
experiments.
2.5
Attachment of the aptamer to ppUCNPs
The obtained ppUCNPs were modied by the aptamer labeled
with 6-carboxytetramethylrhodamine (TAMRA) at the 30 end
(TAMRA-aptamer). 2 mg ppUCNPs were diluted with 2 mL Tris–
HCl buffer (20 mM, containing 10 mM NaCl and 10 mM MgCl2,
pH 7.4) and incubated with TAMRA-aptamer (1 mM) at 30 C for
30 min. The ppUCNPs attached with the aptamer (ppUCNPsaptamer) were harvested with centrifugation and washing.
Finally, the product was diluted with 2 mL Tris–HCl buffer
(20 mM, containing 10 mM NaCl and 10 mM MgCl2, pH 7.4) and
stored at 4 C for further use.
2.6
Upconversion uorescence experiments
In a typical UCNPs-based FRET assay procedure, the ppUCNPsaptamer (200 mL) and various concentrations of lysozyme or the
target DNA were mixed in tubes. Aer adjusting the total
volume to 600 mL with the buffer, the system was incubated at
30 C for 80 min. The upconversion luminescence (UCL) spectra
of the nal mixture were recorded on a ZolixScan ZLX-UPL
spectrometer with an external 980 nm laser as the excitation
source. To assess the specicity of the sensor, four other
biomolecules including a-amylase, bovine serum albumin
(BSA), thrombin and glycine (Gly) were added to the system
following an identical procedure. To verify the ability of the
method to resist background interference, lysozyme detection
was performed in human saliva and serum samples.
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Journal of Materials Chemistry B
Paper
Human saliva samples were collected from 3 healthy
volunteers in the morning before breakfast. The samples were
recollected without the precipitate. 40 mL of the saliva sample
solution and 200 mL of the ppUCNPs-aptamers were mixed in
tubes, and the total volume was adjusted to 600 mL with the
buffer. The recovery experiment was carried out by adding 30
nM of standard lysozyme solution to the system. Aer 80 min of
incubation at 30 C, the system was taken for the UCL
measurement. Human serum mixture samples were collected
from 100 healthy adults in the Affiliated Drum Tower Hospital
of Nanjing University according to their standard clinical
procedures. The determination process was similar to that of
saliva except that 100 mL of the serum samples instead of saliva
samples were used.
2.7
Characterization
The morphology and structure were characterized by transmission electron microscopy (TEM) using a JEOL-2100 TEM
operating at 200 kV. Energy-dispersive X-ray (EDX) analysis
spectra were obtained by a eld-emission scanning electron
microscope (Hitachi S4800, Japan) equipped with an EDAX
Genesis analysis system. X-ray powder diffraction (XRD)
measurements were performed on a Japan Shimadzu XRD-6000
diffractometer with Cu-Ka radiation (l ¼ 0.15418 nm), and a
scanning rate of 0.05 deg s1 was applied to record the patterns
in the 2q range of 10–80 . Zeta potential was measured on a
Nano-Z Zetasizer. The UV-vis absorption and the Fourier
transform infrared (FT-IR) spectra were obtained using a UV3600 spectrophotometer and a NICOLET iS10 FT-IR spectrometer, respectively. Upconversion uorescence spectra were
recorded on a ZolixScan ZLX-UPL spectrometer using an
external 1 W continuous-wave laser (980 nm) as the excitation
source.
3.
3.1
Results and discussion
Scheme 1 Schematic illustration of the FRET biosensor for lysozyme
and DNA detection based on UCNPs.
selected as the dye molecule labeled on the aptamer (abbreviated as TAMRA-aptamer).
In Fig. 1, we can observe that the UV-vis absorption spectrum
of TAMRA-aptamer (acceptor) overlaps well with the uorescence emission band centered at 520 and 543 nm of the
UCNPs (donors), which can be attributed to the 2H11/2 / 4I15/2
and 4S3/2/ 4I15/2 transitions of Er3+, respectively. Meanwhile,
red emission at around 655 nm, assigned to the 4F9/2 / 4I15/2
transition of Er3+,35 should remain without change since it is far
away from the absorption band of TAMRA. To avoid possible
interference, UCL at 655 nm can be taken as a reference to the
green emission to allow ratiometric detection. With static
electricity interaction, TAMRA-aptamer can be brought in close
proximity to the ppUCNPs and the FRET from UCNPs to TAMRA
occurs under the excitation at 980 nm. Upon the addition of
lysozyme to the system, the TAMRA-aptamer was far away from
the ppUCNPs due to the high affinity and specicity between
Principle of the FRET aptasensor for lysozyme detection
A new platform for lysozyme and the target DNA detection was
developed with positive upconversion nanoparticles (ppUCNPs)
and a dye labeled aptamer as the FRET pair. As shown in
Scheme 1, oleic acid capped UCNPs (OA-UCNPs) were rst
synthesized using a hydrothermal route, and the as-prepared
materials could be well dispersed into nonpolar organic
solvents (toluene, hexane, chloroform etc.) to form stable
colloids due to the presence of the hydrophobic oleate anion.
Bio-applications are usually carried out in aqueous solution,
therefore proper dispersion of the nanoparticles in water was
required. To this end, two surface modication steps were
performed on the as-prepared OA-UCNPs. PAA which served as a
multidentate ligand was rst chosen to exchange the original
hydrophobic ligands on the surface of UCNPs. To attach the
negatively charged DNA, an additional layer of PAH was coated
onto the PAA-UCNPs surface, and positively charged ppUCNPs
were obtained. The sensor was set up by coating the dye labeled
aptamer on the surface of the ppUCNPs, and TAMRA was
460 | J. Mater. Chem. B, 2015, 3, 458–464
Fig. 1 The absorption (dot line) and emission (short dash line) spectra
of TAMRA-aptamer aqueous solution besides the emission spectrum
(solid line) of the UCNPs.
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the aptamer and lysozyme. As a result, the luminescence of the
ppUPCNs was restored. Similarly, when the target DNA was
introduced into the system, a DNA duplex structure (dsDNA)
forms due to the hybridization between the aptamer and the
target DNA. As a result, the distance between the ppUCNPs and
TAMRA-aptamer increased and the UCL was restored. The
results showed that the relative UCL intensity was related to the
concentration of lysozyme or the target DNA.
3.2
Characterization of the materials
NaYF4:Yb,Er was selected as the upconversion donor since it
has so far been the most efficient NIR-to-visible upconversion
material due to the low phonon energy of NaYF4 as the host
matrix.5 Extremely strong green emission was obtained and only
a slight uorescence change could be observed aer PAA and
PAH modication (Fig. S1†), so it has no inuence on luminescence measurement. Fig. 2 presents representative TEM
images of the as-prepared and modied upconversion particles.
The monodisperse nanospheres of the as-prepared samples
have an average size of about 29.0 nm (Fig. 2A and D) and the
size changed little aer the two step modication (Fig. 2B and
C). The TEM images also indicated that the as-prepared nanoparticles (OA-UCNPs) were well dispersed into nonpolar
solvents (cyclohexane, chloroform for example) and the modied ones (PAA-UCNPs and ppUCNPs) could be easily dispersed
into water. EDX analysis conrmed the presence of Na, F, Y, Yb
and Er in the three samples and no obvious changes in the
composition of NaYF4, Yb, Er were observed (Fig. S2†). XRD
patterns of OA-UCNPs and PAA-UCNPs reveal that both samples
have similar phases, which can be indexed to the pure hexagonal phase of NaYF4 (JCPDS no. 28-1192) (Fig. 3A).
The capping ligands on the surface of the nanoparticles were
identied by FT-IR spectra (Fig. 3B). All the three samples
exhibit a broad band around 3450 cm1 which corresponds to
Fig. 2 TEM images of OA-UCNPs (A, in cyclohexane), PAA-UCNPs (B,
in water), ppUCNPs (C, in water), and the histogram of particle size for
OA-UCNPs, data were obtained from the TEM images of more than
300 upconversion particles with R-squared value 0.94 (D).
This journal is © The Royal Society of Chemistry 2015
Fig. 3 XRD pattern (A) and FT-IR spectra (B) of the as-prepared and
modified UCNPs.
the O–H stretching vibration. The peaks at 2924 and 2856 cm1
are assigned to the asymmetric (nas) and symmetric (ns)
stretching vibrations of methylene (–CH2–) in the long alkyl
chain of oleic acid; these two peaks become unconspicuous
aer the modication. The peak at 3003 cm1 attributed to the
group ]CH2 can be clearly seen in the spectrum of OA-UCNPs,36
while it disappears aer the modication. In addition, the
peaks at 1558 and 1462 cm1 can be assigned to the asymmetric
(nas) and symmetric (ns) stretching vibrations of the carboxylic
group in oleic acid. Aer ligand exchange by PAA, the two peaks
shi a little and two new peaks at 1732 and 1396 cm1 related to
the carboxylic group appear, indicating that the primary ligand
oleic acid was successfully exchanged by PAA. Upon the
attachment of PAH, the peak at 1732 decreased and a weak peak
at 740 cm1 resulting from the bending vibration of the N–H
bond in PAH could be found,17 which conrmed that ppUCNPs
were formed nally. The process was also veried by zeta
potential spectroscopy. The zeta potential of the nanocomposites changed from negative (37.8 mV) to positive
(+37.4 mV) alternated aer PAA and PAH modication, while
the potential was found to be 9.19 mV aer the addition of
TAMRA-aptamer (1 mM). The zeta results further indicated that
the process was realized as expected.
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3.3 FRET between ppUCNPs and TAMRA induced through
the aptamer
In order to attach more TAMRA-aptamer and obtain high FRET
efficiency, the excess of the unbounded cationic polymer PAH in
the positive UCNPs solution had to be minimized. An experiment was carried out by gradually adding PAH to a solution
containing PAA-UCNPs, and zeta potential was measured
(Fig. 4A), the results showed that the zeta potential reached its
maximum at the concentration of 0.75 mg mL1. As shown in
Fig. 4B, when lysozyme was added to the ppUCNPs-aptamer
solution, the UCL intensity increased slowly within 80 min
incubation. Aer that, the intensity was virtually unchanged. So
UCL spectra of the sensor with various concentrations of
lysozyme, inset: amplified UCL spectra around 580 nm (A). The linear
relationship of the relative fluorescence intensity versus the concentration of lysozyme in the range from 30 to 210 nM; inset: fluorescence
recovery of the sensor in the presence of 0–300 nM lysozyme. I0 and I
denote the relative fluorescence intensity UCL510–565/UCL640–680
before and after addition of lysozyme (B).
Fig. 5
Fig. 4 Zeta potential changes with the addition of PAH (A). Time
dependence of the fluorescence recovery of the ppUCNPs-aptamer
complex upon the addition of 300 nM lysozyme; inset: the green to
red ratio (GRR, UCL510–565/UCL640–680) of UCL intensity at different
times (B). The green to red ratio of UCL intensity with different lysozyme concentrations before and after PAH modification (C).
462 | J. Mater. Chem. B, 2015, 3, 458–464
Fig. 6 Specificity of the aptamer-ppUCNPs toward other molecules.
Mixture represents the mixture of lysozyme and the other four
molecules. The concentration of a-amylase and Gly was 15.0 mg L1
and 3 mM respectively, the concentration of the other three molecules
was 300 nM.
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Table 1
Journal of Materials Chemistry B
Lysozyme recovery in diluted human saliva (n ¼ 4)
Sample
Measured (nM)
Added (nM)
Found (nM)
Recovery (%)
RSD (%)
1
2
3
95.3
143.1
121.0
30
30
30
114.2
179.1
143.7
91.1
103.5
95.2
3.3
2.1
2.6
0.75 mg mL1 PAH was needed and the UCL measurement was
carried out aer incubating for 80 min. To conrm the
adsorption of PAH, a control experiment was conducted where
PAA-UCNPs (without PAH modication) at 0.2 mg mL1 with
different concentrations of lysozyme were incubated with
TAMRA-aptamer (1 mM). In this case, the UCL intensity was
restored a little and the green to red ratio (GRR, UCL510–565/
UCL640–680, UCLA–B are the integrated emission intensities from
A to B nm) changed slightly compared with that using ppUCNPs
(Fig. 4C).
3.4
Analysis of lysozyme and the target DNA
The UCL of the UPCNs was quenched with the addition of
TAMRA-aptamer (1 mM). When increasing amounts of lysozyme
were introduced into the system, the emission intensity of the
UCNPs was restored gradually. This can be explained by the
separation of the acceptor from the donor because the TAMRAaptamer prefers combining with lysozyme to ppUCNPs. The
relative uorescence intensity was linearly related to the
concentration of lysozyme ranging from 30 to 210 nM (Fig. 5)
and the limit of detection (LOD) of lysozyme is as low as 2.8 nM
calculated by 3SD/m, where 3 is the factor at the 99% condence
level, SD represents the standard deviation of the blank
measurements (n ¼ 8) and m is the slope for the range of linearity.15,37 The sensor can also be used in DNA determination,
and the relative uorescence intensity was also linearly related
to the concentration of the target DNA in the range of 40 to 200
nM with the LOD of 2.8 nM (Fig. S3†).
3.5
relative standard deviation (RSD) around 3% (Table 1), indicating a high level of accuracy of the developed assay. To
broaden the application, the concentrations of lysozyme in
human serum were also measured, and the results shown in
Table S2† were within the normal range of the reported literature.39,40 The results indicate that the developed sensor is
applicable in such a complicated matrix, which shows its
potential use in biological and clinical applications.
4. Conclusions
In summary, we have fabricated a novel upconversion FRET
sensing platform for the determination of lysozyme as well as
DNA. The energy transfer occurs between the upconversion
nanoparticles and the dye labeled aptamer, and a cationic
polymer that acted as an electrostatic linker was used to bring
the donor and acceptor into close proximity. However, in the
presence of lysozyme or the complementary DNA, uorescence
recovery was observed, and the relative recovery intensity was
proportional to its concentration. The method shows high
sensitivity as well as good selectivity, and because it is a labelfree method without covalent linking, the assay is fairly simple
and convenient. Besides, UCNPs as excellent emitters with their
low autouorescence and high penetration depth endow this
system with further potential applications in biological and
analytical elds. Therefore, we believe that this general method
can be extended to numerous other uorescent sensing probes
and may be put into practice in determining lysozyme in human
samples.
Specicity evaluation and analytical application
To examine the specicity of the sensor for lysozyme, control
experiments were performed with the xed concentration of
aptamer-ppUCNPs in the presence of a-amylase, BSA, thrombin
and Gly, respectively. In addition, a mixture of lysozyme and the
four non-specic biomolecules was also tested. As shown in
Fig. 6, obvious response was observed when lysozyme or the
mixture of lysozyme and the four non-specic biomolecules was
added to the assay system, whereas the non-specic biomolecules gave little response. The results show the high selectivity
of this method.
The method was applied to detect lysozyme levels in human
saliva samples. Taking the dilution into account in the calculation, the obtained values are between 1.4 and 2.1 mM (Table
S1†), which are consistent with the normal range of the reported
literature.31,38 To further validate the developed method, 30 nM
of lysozyme was added to the diluted saliva samples. The
recoveries were in the range from 91.1% to 103.5% with a
This journal is © The Royal Society of Chemistry 2015
Acknowledgements
We greatly appreciate the nancial support from the National
Natural Science Foundation of China (NSFC) (no. 21171091,
21335004, 21405001) and the support from 973 Program (no.
2011CB933502). The authors extend their appreciation to the
State Key Laboratory of Analytical Chemistry for Life science
(SKLACLS1307).
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